1. Field of the Invention
The present invention relates to antioxidants, and particularly to neuroprotective multifunctional antioxidants that can both chelate metals, such as Fe, Cu or Zn, and scavenge free radicals. The invention also relates to antioxidant monofunctional analogs of the neuroprotective multifunctional antioxidant compounds. The compounds can be administered orally, and can cross the blood-brain barrier, so that the compounds are beneficial for the treatment of various neurological disorders, such as Alzheimer's disease, Parkinson's disease, ALS (amyotrophic lateral sclerosis), traumatic brain injury, ocular disorders such as cataract, glaucoma, age-related macular degeneration and other retinal degeneration, as well as reducing the progression of diabetic complications.
2. Description of the Related Art
Oxidative damage is a hallmark of neurodegenerative disorders. Oxidative stress results from reactive oxygen species (ROS) that damage cellular components by oxidizing proteins, lipid bilayers and DNA. This results in altered protein conformations, reduced enzyme activities, lipid peroxide generation that disrupts plasma and organelle membranes, and altered DNA, which leads to strand breaks, DNA-protein cross-linking, and mutations through base modifications. ROS includes the super oxide anion (O2−), the hydroxyl radical (—OH), singlet oxygen (1O2), and hydrogen peroxide (H2O2). Superoxide anions continuously form in mitochondria, as molecular oxygen (O2) acquires an additional electron. Hydroxyl radicals, the most reactive and damaging of generated ROS, predominantly form by a Fenton reaction between hydrogen peroxide and redox active transition metals, such as iron and copper. Although these metals are oxidized during this process, they are returned to their “active” (reduced) state through a process of “redox cycling” with vitamin C or other cellular reductants. Hydrogen peroxide, produced in vivo through several reactions, can either be converted to the highly reactive and damaging hydroxyl radicals, or converted to water It is formed by the reduction of superoxide radical by superoxide dismutase and reduced to water by either catalase or glutathione peroxidase.
Oxidative stress increases with age, and prolonged exposure of tissues to oxidative stress results in cellular damage that eventually leads to cell death. ROS activity has been observed in the hippocampus, substantia nigra and caudate putamen of the brain and in the spinal fluid. Neural tissues in the brain are especially susceptible to ROS because of the higher metabolic rates, high compositions of peroxidation susceptible fatty acids, high intracellular concentrations of transition metals capable of catalyzing Fenton reactions, low levels of antioxidants, and reduced capability for tissue regeneration. Neural tissues also possess brain-specific oxidases, such as monoamine oxidase, that can generate hydrogen peroxide. Neuroinflammatory responses induced by reactive microglia, macrophages and proinflammatory T-cells can also generate ROS.
In the brain, the redox-active metals iron (Fe), copper (Cu), and zinc (Zn) accumulate with age, and this accumulation is linked to altered brain metabolism and increased amyloid precursor protein (APP) expression. Amyloid beta (Aβ) is the major proinflammatory component of Alzheimer's disease (AD) plaques, and its binding to Cu, Fe, and Zn promotes Aβ aggregation into protease-resistant, metal-enriched precipitates. Aβ efficiently generates reactive oxygen species in the presence of copper and iron. Aberrant biometal homeostasis and metalloprotein reactions occur during the development of AD and results in oxidation-linked neurodegeneration.
The age-dependent accumulation of Fe also alters Fe metabolism in the brain in AD and Parkinson's disease (PD), which has been linked to changes in the expression of lactotransferrin receptor, melanotransferrin, ceruloplasmin and divalent cation transporters in brain ion transport. Increased Fe levels have also been observed in pathologically affected areas of postmortem brains in other neurodegenerative diseases, such as Parkinson's patients, and these areas correspond to an increased severity of neuropathological changes. Changes in Cu levels can also affect the brain by interfering with Fe. The Cu-binding enzyme ceruloplasmin represents a link between Cu and Fe metabolism because this enzyme regulates the Fe redox state through its ferroxidase activity by converting Fe (II) to Fe (III). Ceruloplasmin is rapidly degraded when Cu is not properly incorporated into the protein at the rate of the protein synthesis, as seen in aceruloplasminemia, where altered Fe hemostasis occurs with marked Fe accumulation into neuroglia and neurons. In AD patients, a decreased neuronal induction of ceruloplasmin may lead to an accumulation of redox-active iron in neurons.
Targeting oxidative pathways associated with neurodegeneration can be therapeutic. Reducing ROS with free radical scavenging antioxidants ranging from natural products (curcumin, melatonin, resveratrol, Ginkgo biloba extract, green tea, vitamin C, L-carnitine, vitamin E, and cannabinoids) to lipoic acid derivatives, Coenzyme Q (MitoQ) analogs, and “thiol-delivering” glutathione-mimics have been reported. However, the ability of most of these compounds to cross the blood-brain barrier (BBB) has not been demonstrated.
ROS can also be reduced through the use of biometal attenuating compounds. Desferoxamine (desferrioxamine) can bind Fe, Cu, and Zn and decrease AD progression. However, desferoxamine is not orally active and does not significantly cross the BBB. DdP109, a more lipophilic chelator, has been reported to reduce the levels of aggregated insoluble Aβ and increase its soluble forms when administered to transgenic mice. The orally active metal chelator clioquinol (PBT1), which modulates the Fenton reaction, decreases Cu uptake in the brain, disaggregates redox metal-induced Aβ aggregation, and retards fibril growth, shows efficacy in both animals and several clinical trials. Oral PBT2 also reduces Aβ aggregation and toxicity by interfering with the redox activity associated with Aβ-metal complexes. PBT2 significantly reduces Aβ concentrations in the brain and rapidly reversed cognitive deficits as demonstrated in a Phase IIa clinical trial, where AD patients improved in two neuropsychological tests. These results support the premise that attenuation of metal-protein interactions is a promising strategy for therapeutic intervention.
Iron can be released from hemoglobin during traumatic brain injury (TBI) or hemorrhagic stroke. The increase in free ferrous iron can lead to oxidative damage. Children are especially vulnerable to TBI induced hemorrhage and cell death because their immature brain has a muted response to oxidative stress due to inadequate expression of certain antioxidant molecules, and their developing brain is less able to detoxify free iron. TBI also elicits an acute inflammatory response in which ROS is generated. Anti-inflammatory agents, antioxidants, and the iron chelator desferoxamine have been proposed to treat the increased inflammation, oxidative stress and presence of free iron levels observed in adult and pediatric TBI.
Age-related macular degeneration (AMD) risk factors, such as smoking, suggest that AMD is linked to oxidative stress. A role for oxidative stress in AMD is supported by the AREDS trial results, which found that antioxidants and zinc reduce the risk of AMD progression, as well as oxidative stress-induced endothelial dysfunction, by reducing ROS. Patients with AMD have higher retinal levels of lipid peroxidation products, which are present in drusen. The retina is vulnerable to oxidative stress because of its high levels of oxygen that are required for retinal function. Moreover, the membranes of rods and cones in the outer nuclear layer (ONL) contain a high percentage of polyunsaturated fatty acids that are susceptible to lipid peroxidation. The macular region is particularly susceptible to ROS because incoming light is focused onto the macula. Incoming light is a constant source of oxidative stress because photo-oxidation generates ROS. ROS is also generated by retinal pigmented epithelial (RPE) cell phagocytosis and the photosensitizing activity of lipofuscin. Exposing RPE cells to ROS leads to apoptosis and premature senescence
Aβ deposition is also present in AMD. Drusen, a biomarker for AMD that forms adjacent to the RPE, contains Aβ, whose presence has been linked to local inflammatory events. As the major pro-inflammatory component of AD plaques, retinal Aβ has been linked to RPE dysfunction that results in retinal degeneration and AMD. In RPE, Aβ accumulation also affects the balance between vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF).
Several animal models show AMD-like retinal changes that are linked to oxidative stress, iron dysregulation, and Fenton chemistry. These include the C57BL/6 mice exposed to cigarette smoke, mice deficient in SOD1, knockout mice (DKO) deficient in ceruloplasmin (Cp) and its homolog hephaestin, and RCS rats. Introducing iron directly into the eye also produces retinal degeneration.
Targeting oxidative pathways associated with AMD-linked retinal changes shows therapeutic potential. The AREDS antioxidant formulation reduces the risk of AMD progression to the advanced stage by 25%. Antioxidants also reduce oxidative stress in cultured RPE cells. Addition of bovine melanosomes or melatonin to non-pigmented bovine RPE also reduces the photosensitized and iron-mediated oxidation of RPE cells. The antioxidant N-tert-butyl hydroxylamine (NtBHA), when added to iron-overloaded human RPE, reduces ROS and maintains GSH levels. Similarly, treatment with salicylaldehyde isonicotinyl hydrazone (SIH) protects RPE cells against the Fenton-generated hydroxyl radicals. The antioxidant quercetin also protects RPE against hydrogen peroxide-induced oxidative stress. Free radical scavengers, such as N-acetylcysteine, dimethylthiourea, Ginkgo biloba extract, phenyl-N-tert-butylnitrone, WR-77913, Tempol H, and edaravone, protect against light-induced retinal degeneration. Retinal degeneration in DKO mice is reduced with the iron chelator deferiprone. Administration of the multifunctional antioxidant JHX-4 also protects rats against light-induced retinal damage by reducing biomarkers of oxidative stress in the neural retinas, preservation of retinal ERG patterns, and preservation of the photoreceptor layer.
In addition to AMD, iron-associated oxidative injury plays a role in retinal degenerations, such as retinitis pigmentosa. Zinc-deferoxamine has been known to attenuate retinal degeneration in the rd10 mouse model of retinitis pigmentosa.
Increased mRNA and protein levels for the iron-regulating proteins transferrin, ceruloplasmin, and ferritin are present in glaucoma. By inducing lysosomal membrane permeabilization and the release of cathepsin D into the cytosol, ROS leads to trabecular meshwork (TM) cell death. This cell death is reduced by chelation. Lysosomes degrade organelles, long-lived proteins, and extracellular and membrane-bound materials. Significant concentrations of labile iron can accumulate within lysosomes because of their breakdown of iron-containing endocytosed and autophagocytosed materials. This can result in lysosomal hydroxyl radicals being generated through Fenton reactions. Neuroprotection by iron chelators prevents hydroxyl radical formation in the Fenton reaction by sequestering redox-active iron. Iron chelators can also upregulate or stabilize hypoxia-inducible factor-1a (HIF-1a). The stability of HIF-1a is controlled by iron-dependent oxygen-sensor enzymes, HIF prolyl-4-hydroxylases (PHDs) that target HIF-1a for degradation. HIF-1a is present in the glaucomatous retina and has been linked to RGC death. The HIF system is an emerging target for neuroprotection because it promotes the stabilization of bHIF-1a and increases transcription of HIF-1-related survival genes. Iron chelators appear to provide neuroprotection by inhibiting PHDs that target the HI″F-1 signaling pathway and ultimately activate the HIF-1-dependent neuroprotective genes.
Cataracts linked to oxidative stress and ROS include those associated with aging, ionizing and UV radiation, increased oxygen tension resulting from vitrectomy surgery, and tobacco smoke. In many of these cataracts, the Fenton reaction contributes to ROS because Cu and Fe also accumulate in lenses with aging and exposure to tobacco smoke. In AD patients, Aβ deposition also causes cataracts by accumulating as electron-dense deposits in the cytoplasm of supranuclear/deep cortical lens fibers cells. Aβ deposits similarly occur in Aβ transgenic mice, where they can be reduced by chelation.
In cataracts, antioxidants have been widely used to reduce cataract formation in experimental animals. Chelation reduces Aβ deposition observed clinically and experimentally in mice. Chelation also reduces cataracts in β-thalassemia patients, and in tobacco smoke-exposed rats.
Cellular exposure to ionizing radiation can alter atomic structures through either direct interactions of the radiation with target macromolecules, or indirectly through the generation of ROS by water radiolysis. Moreover, the oxidative damage may spread from the targeted to neighboring, non-targeted bystander cells through redox-modulated intercellular mechanisms. Radiation can also initiate the release of iron via the photoreduction of iron stored inside ferritin.
Chelation of Cu, Fe, Mn, and Zn facilitates the tissue repair processes required for recovery from radiation injury, including survival of lethally irradiated mice and rats. Iron chelators may also help prevent photo-aging. Administration of the multifunctional antioxidant JHX-4 to Long-Evans rats that were administered 15 Gy of whole head gamma irradiation significantly delayed cataract formation, in addition to partially alleviating a reduction in weight loss due to apparent salivary gland response to irradiation.
Oxidative stress is also one of the major causative factors for diabetes and diabetic complications, and increased heme iron has been significantly associated with an increased risk of insulin resistance and type-2 diabetes. Experimentally, antioxidants and chelators have been observed to be beneficial in the treatment of nerve and vascular dysfunction in experimental diabetes. Administration of the multifunctional antioxidant JHX-4 to diabetic rats also delayed the progression of cataracts.
Thus, neuroprotective multifunctional antioxidants solving the aforementioned problems is desired.